WO2012174337A1 - Procédés de préparation de clonage in vitro - Google Patents

Procédés de préparation de clonage in vitro Download PDF

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Publication number
WO2012174337A1
WO2012174337A1 PCT/US2012/042597 US2012042597W WO2012174337A1 WO 2012174337 A1 WO2012174337 A1 WO 2012174337A1 US 2012042597 W US2012042597 W US 2012042597W WO 2012174337 A1 WO2012174337 A1 WO 2012174337A1
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WIPO (PCT)
Prior art keywords
nucleic acid
oligonucleotide
molecules
acid molecules
sequence
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PCT/US2012/042597
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English (en)
Inventor
Li-Yun A. KUNG
Daniel Schindler
Joseph Jacobson
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Gen9, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gen9, Inc. filed Critical Gen9, Inc.
Priority to EP12735365.4A priority Critical patent/EP2721154B1/fr
Priority to CN201280039777.8A priority patent/CN103732744A/zh
Priority to LTEP12735365.4T priority patent/LT2721154T/lt
Priority to AU2012271487A priority patent/AU2012271487B2/en
Priority to CA2839418A priority patent/CA2839418A1/fr
Priority to EP18207413.8A priority patent/EP3517611A3/fr
Publication of WO2012174337A1 publication Critical patent/WO2012174337A1/fr
Priority to IL230009A priority patent/IL230009B/en
Priority to IL287337A priority patent/IL287337A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/191—Modifications characterised by incorporating an adaptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2533/00—Reactions characterised by the enzymatic reaction principle used
    • C12Q2533/10—Reactions characterised by the enzymatic reaction principle used the purpose being to increase the length of an oligonucleotide strand
    • C12Q2533/107—Probe or oligonucleotide ligation

Definitions

  • Methods relate to the selection of a target nucleic acid sequence from a population of nucleic acid sequences. More particularly methods are provided for isolation of target sequences nucleic acid sequences of interest away from a library of nucleic acid sequences.
  • transformation which typically confer an antibiotic resistance to the strain.
  • Selection of cells of the strain and proper dilution of the cells onto agar plates allows for identification of colonies arising from individual bacterial cells carrying individual sequences from the initial mixture. Subsequent manipulation of these colonies allow for the identification of target sequences away from the non-target sequences in the pool.
  • the limiting dilution technique has been used to isolate single cells from a pooled suspension of cells, as well as for counting of DNA molecules.
  • the basic method involves making a measurement of the number of cells or the concentration of DNA, diluting to very low concentration, and aliquoting into separate wells such that the number of cells or DNA molecules is less than one per well.
  • Subsequent cell division or DNA amplification e.g. by PCR should reveal a Poissonian distribution of the wells corresponding to wells which were initially empty, seeded with one cell or molecule, and those seeded with multiples.
  • the method comprises the steps of providing a population of nucleic acid molecules comprising a plurality of distinct nucleic acid molecules; providing a plurality of oligonucleotide tags; attaching the oligonucleotide tags to one end terminus of the plurality nucleic acid molecules thereby generating a subpopulation of nucleic acid-oligonucleotide tag molecules; amplifying the plurality of nucleic acid- oligonucleotide tag molecules using primers complementary to the plurality of oligonucleotide tags; and isolating at least one target nucleic acid-oligonucleotide tag molecule of the
  • the method further comprises sequencing the at least one isolated target nucleic acid-oligonucleotide tag molecule and identifying the target nucleic acid. In some embodiments, the sequencing step is by high throughput sequencing. In some embodiments, the method further comprises amplifying the at least one target nucleic acid- oligonucleotide tag molecule.
  • the step of isolating is by limiting dilution.
  • the target nucleic acid-oligonucleotide tag molecule is amplified.
  • Some aspects of the invention relate to a method of isolating a target nucleic acid, comprising (a) providing a population comprising a plurality of different nucleic acid molecules;
  • each microparticle has an oligonucleotide sequence complementary to a portion of the nucleic acid molecules immobilized on its surface;
  • each microparticle has a single complementary sequence on its surface.
  • the complementary oligonucleotide sequences are identical. Yet in other embodiments, the complementary oligonucleotide sequences are different.
  • the plurality of nucleic acid molecules comprises an oligonucleotide tag at one terminus. In some embodiments, the plurality of nucleic acid molecules can have the same oligonucleotide tag or a different oligonucleotide tag. In some embodiments, the step of isolating is by limiting dilution. In some embodiments, the method further comprises amplifying the at least one target molecule.
  • Some aspects of the invention relate to a method of isolating a target nucleic acid, the method comprising (a) providing a population comprising a plurality of different nucleic acid molecules; (b) providing a dilution of the population of nucleic acid molecules; (c) separating the nucleic acid molecules into samples comprising a single molecule or a smaller number of molecules; (d ) amplifying the single molecules thereby forming amplified nucleic acid molecules; (e) optionally repeating step (c) and (d) in samples that do not comprise a nucleic acid molecule; and (f) isolating at least one target nucleic acid molecule.
  • the target nucleic acid molecule has a predefined sequence.
  • the method further comprises sequencing the at least one target nucleic acid molecule.
  • FIG. 1 illustrates a non- limiting exemplary method of in vitro cloning by increasing the limiting dilution.
  • Fig. 2 illustrates a non-limiting exemplary method of in vitro cloning using beads with single ligand sites for nucleic acid attachment.
  • Fig. 3 illustrates a non- limiting exemplary method of in vitro cloning using a library of barcode sequences.
  • aspects of the invention relate to methods and compositions for analyzing and separating at least one nucleic acid having a predefined sequence from a pool comprising a plurality of different nucleic acid sequences.
  • the pool of nucleic acid sequences comprises variants of the nucleic acid sequence of interest and/or nucleic acid sequences having a similar length than the nucleic acid sequence of interest.
  • aspects of the invention is particularly useful for isolating nucleic acids sequence of interest from a population of nucleic acid sequences such as a library of nucleic acid sequences.
  • nucleic acid isolating nucleic acid sequences for assembly of nucleic acid molecules having a predefined sequence.
  • nucleic acid polynucleotide
  • oligonucleotide are used interchangeably and refer to naturally-occurring or synthetic polymeric forms of nucleotides.
  • oligonucleotides and nucleic acid molecules of the present invention may be formed from naturally occurring nucleotides, for example forming deoxyribonucleic acid (DNA) or ribonucleic acid (R A) molecules.
  • the naturally occurring oligonucleotides may include structural modifications to alter their properties, such as in peptide nucleic acids (PNA) or in locked nucleic acids (LNA).
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • Nucleotides useful in the invention include, for example, naturally-occurring nucleotides (for example, ribonucleotides or deoxyribonucleotides), or natural or synthetic modifications of nucleotides, or artificial bases.
  • monomer refers to a member of a set of small molecules which are and can be joined together to from an oligomer, a polymer or a compound composed of two or more members.
  • the particular ordering of monomers within a polymer is referred to herein as the "sequence" of the polymer.
  • the set of monomers includes but is not limited to example, the set of common L-amino acids, the set of D- amino acids, the set of synthetic and/or natural amino acids, the set of nucleotides and the set of pentoses and hexoses.
  • predetermined sequence or “predefined sequence” are used interchangeably and means that the sequence of the polymer is known and chosen before synthesis or assembly of the polymer.
  • aspects of the invention is described herein primarily with regard to the preparation of nucleic acids molecules, the sequence of the oligonucleotide or polynucleotide being known and chosen before the synthesis or assembly of the nucleic acid molecules.
  • immobilized oligonucleotides or polynucleotides are used as a source of material.
  • oligonucleotides their sequence being determined based on the sequence of the final polynucleotides constructs to be synthesized.
  • oligonucleotides are short nucleic acid molecules.
  • oligonucleotides may be from 10 to about 300 nucleotides, from 20 to about 400 nucleotides, from 30 to about 500 nucleotides, from 40 to about 600 nucleotides, or more than about 600 nucleotides long. However, shorter or longer oligonucleotides may be used. Oligonucleotides may be designed to have different lengths. In some embodiments, the sequence of the polynucleotide construct may be divided up into a plurality of shorter sequences that can be synthesized in parallel and assembled into a single or a plurality of desired polynucleotide constructs using the methods described herein.
  • the assembly procedure may include several parallel and/or sequential reaction steps in which a plurality of different nucleic acids or oligonucleotides are synthesized or immobilized, primer-extended, and are combined in order to be assembled (e.g., by extension or ligation as described herein) to generate a longer nucleic acid product to be used for further assembly, cloning, or other applications.
  • the nucleic acids molecules prepared according to the methods disclosed herein can be used for nucleic acid assembly and for assembly of libraries containing nucleic acids having predetermined sequence variations. Assembly strategies provided herein can be used to generate very large libraries representative of many different nucleic acid sequences of interest.
  • libraries of nucleic acids are libraries of sequence variants. Sequence variants may be variants of a single naturally-occurring protein encoding sequence. However, in some embodiments, sequence variants may be variants of a plurality of different protein-encoding sequences. Accordingly, one aspect of the technology provided herein relates to the assembling of precise high-density nucleic acid libraries.
  • a high-density nucleic acid library may include more that 100 different sequence variants (e.g., about 10 2 to 10 3 ; about 10 3 to 10 4 ; about 10 4 to 10 5 ; about 10 5 to 10 6 ; about 10 6 to 10 7 ; about 10 7 to 10 8 ; about 10 8 to 10 9 ; about 10 9 to 10 10 ; about 10 10 to 10 11 ; about 10 11 to 10 12 ; about 10 12 to 10 13 ; about 10 13 to 10 14 ; about 10 14 to 10 15 ; or more different sequences) wherein a high percentage of the different sequences are specified sequences as opposed to random sequences (e.g., more than about 50%, more than about 60%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%
  • sequence variants e.g., about 10 2 to 10 3 ; about 10 3
  • oligonucleotides that are immobilized on a surface or substrate (e.g., support-bound
  • Support-bound oligonucleotides comprise for example, oligonucleotides complementary to construction oligonucleotides, anchor oligonucleotides and/or spacer oligonucleotides.
  • support refers to a porous or non-porous solvent insoluble material on which polymers such as nucleic acids are synthesized or immobilized.
  • porous means that the material contains pores having substantially uniform diameters (for example in the nm range). Porous materials include paper, synthetic filters etc. In such porous materials, the reaction may take place within the pores.
  • the support can have any one of a number of shapes, such as pin, strip, plate, disk, rod, bends, cylindrical structure, particle, including bead, nanoparticles and the like.
  • the support can have variable widths.
  • the support can be hydrophilic or capable of being rendered hydrophilic.
  • the support can include inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF) membrane, glass, controlled pore glass, magnetic controlled pore glass, ceramics, metals, and the like etc.; either used by themselves or in conjunction with other materials.
  • inorganic powders such as silica, magnesium sulfate, and alumina
  • natural polymeric materials particularly cellulos
  • oligonucleotides are synthesized on an array format.
  • single-stranded oligonucleotides are synthesized in situ on a common support wherein each oligonucleotide is synthesized on a separate or discrete feature (or spot) on the substrate.
  • single-stranded oligonucleotides are bound to the surface of the support or feature.
  • array refers to an arrangement of discrete features for storing, amplifying and releasing oligonucleotides or complementary oligonucleotides for further reactions.
  • the support or array is addressable: the support includes two or more discrete addressable features at a particular predetermined location (i.e., an
  • each oligonucleotide molecule of the array is localized to a known and defined location on the support.
  • the sequence of each oligonucleotide can be determined from its position on the support.
  • the array may comprise interfeatures regions. Interfeatures will typically not carry any oligonucleotide on their surface and will correspond to inert space.
  • oligonucleotides are attached, spotted, immobilized, surface-bound, supported or synthesized on the discrete features of the surface or array.
  • Oligonucleotides may be covalently attached to the surface or deposited on the surface.
  • Arrays may be constructed, custom ordered or purchased from a commercial vendor (e.g., Agilent, Affymetrix, Nimblegen).
  • Various methods of construction are well known in the art e.g., maskless array synthesizers, light directed methods utilizing masks, flow channel methods, spotting methods, etc.
  • construction and/or selection oligonucleotides may be synthesized on a solid support using maskless array synthesizer (MAS).
  • MAS maskless array synthesizer
  • Maskless array synthesizers are described, for example, in PCT application No. WO 99/42813 and in
  • oligonucleotides include, for example, light-directed methods utilizing masks, flow channel methods, spotting methods, pin-based methods, and methods utilizing multiple supports.
  • Light directed methods utilizing masks e.g., VLSIPSTM methods
  • VLSIPSTM methods for the synthesis of oligonucleotides is described, for example, in U.S. Pat. Nos. 5,143,854, 5,510,270 and 5,527,681. These methods involve activating predefined regions of a solid support and then contacting the support with a preselected monomer solution. Selected regions can be activated by irradiation with a light source through a mask much in the manner of photolithography techniques used in integrated circuit fabrication.
  • a light pattern defines which regions of the support react with a given monomer.
  • a diverse array of polymers is produced on the support.
  • Other steps such as washing unreacted monomer solution from the support, can be optionally used.
  • Other applicable methods include mechanical techniques such as those described in U.S. Pat. No. 5,384,261. Additional methods applicable to synthesis of oligonucleotides on a single support are described, for example, in U.S. Pat. No. 5,384,261.
  • reagents may be delivered to the support by either (1) flowing within a channel defined on predefined regions or (2) "spotting" on predefined regions. Other approaches, as well as combinations of spotting and flowing, may be employed as well. In each instance, certain activated regions of the support are mechanically separated from other regions when the monomer solutions are delivered to the various reaction sites.
  • Flow channel methods involve, for example, microfluidic systems to control synthesis of oligonucleotides on a solid support.
  • diverse polymer sequences may be synthesized at selected regions of a solid support by forming flow channels on a surface of the support through which appropriate reagents flow or in which appropriate reagents are placed.
  • Spotting methods for preparation of oligonucleotides on a solid support involve delivering reactants in relatively small quantities by directly depositing them in selected regions. In some steps, the entire support surface can be sprayed or otherwise coated with a solution, if it is more efficient to do so. Precisely measured aliquots of monomer solutions may be deposited dropwise by a dispenser that moves from region to region.
  • Pin-based methods for synthesis of oligonucleotides on a solid support are described, for example, in U.S. Pat. No. 5,288,514. Pin-based methods utilize a support having a plurality of pins or other extensions. The pins are each inserted simultaneously into individual reagent containers in a tray.
  • An array of 96 pins is commonly utilized with a 96-container tray, such as a 96-well microtiter dish.
  • a 96-container tray such as a 96-well microtiter dish.
  • Each tray is filled with a particular reagent for coupling in a particular chemical reaction on an individual pin. Accordingly, the trays will often contain different reagents. Since the chemical reactions have been optimized such that each of the reactions can be performed under a relatively similar set of reaction conditions, it becomes possible to conduct multiple chemical coupling steps simultaneously.
  • a plurality of oligonucleotides may be synthesized or immobilized on multiple supports.
  • One example is a bead based synthesis method which is described, for example, in U.S. Pat. Nos. 5,770,358; 5,639,603; and 5,541,061.
  • a suitable carrier such as water
  • the beads are provided with optional spacer molecules having an active site to which is complexed, optionally, a protecting group.
  • the beads are divided for coupling into a plurality of containers.
  • Pre-synthesized oligonucleotide and/or polynucleotide sequences may be attached to a support or synthesized in situ using light-directed methods, flow channel and spotting methods, inkjet methods, pin-based methods and bead-based methods set forth in the following references: McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93: 13555; Synthetic DNA Arrays In Genetic Engineering, Vol. 20: 111, Plenum Press (1998); Duggan et al. (1999) Nat. Genet. S21 : 10 ; Microarrays: Making Them and Using Them In Microarray Bioinformatics, Cambridge University Press, 2003; U.S. Patent Application Publication Nos. 2003/0068633 and
  • pre-synthesized oligonucleotides are attached to a support or are synthesized using a spotting methodology wherein monomers solutions are deposited dropwise by a dispenser that moves from region to region (e.g., inkjet).
  • oligonucleotides are spotted on a support using, for example, a mechanical wave actuated dispenser.
  • the invention relates to a method for producing target
  • polynucleotides having a predefined sequence on a solid support can be at least about 1, 2, 3, 4, 5, 8, 10, 15, 20, 25, 30, 40, 50, 75, or 100 kilobases (kb), or 1 megabase (mb), or longer.
  • the invention relate to a method for the production of high fidelity polynucleotides.
  • a composition of synthetic polynucleotides contains at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 50%, 60%), 70%), 80%), 90%), 95 % or more, copies that are error free (e.g., having a sequence that does not deviate from a predetermined sequence).
  • the percent of error free copies is based on the number of error free copies in the composition as compared to the total number of copies of the polynucleotide in the composition that were intended to have the correct, e.g., predefined or predetermined, sequence.
  • aspects of the invention relate to the preparation of oligonucleotides for high fidelity polynucleotide assembly. Aspects of the invention may be useful to increase the throughput rate of a nucleic acid assembly procedure and/or reduce the number of steps or amounts of reagent used to generate a correctly assembled nucleic acid. In certain embodiments, aspects of the invention may be useful in the context of automated nucleic acid assembly to reduce the time, number of steps, amount of reagents, and other factors required for the assembly of each correct nucleic acid. Accordingly, these and other aspects of the invention may be useful to reduce the cost and time of one or more nucleic acid assembly procedures.
  • sequence of a nucleic acid may include some errors that may result from sequence errors introduced during the oligonucleotides synthesis, the synthesis of nucleic acids, and/or from assembly errors during the assembly reaction.
  • unwanted sequences may be present in some nucleic acids. For example, between 0%> and 50%> (e.g., less than 45%, less than 40%), less than 35%, less than 30%>, less than 25%, less than 20%, less than 15%, less than 10%), less than 5% or less than 1%) of the sequences in a library may be unwanted sequences.
  • nucleic acid having the predefined (or correct sequence) can be selectively isolated.
  • selective isolation as used herein, can involve physical isolation of a.
  • desired polynucleotide from others as by selective physical movement of the desired
  • polynucleotide as well as selective mactivation, destruction, release, or removal of other polynucleotides than the polynucleotide of interest.
  • Devices and methods to selectively isolate the correct nucleic acid sequence from the incorrect nucleic acid sequences form a pool of nucleic acid sequences are provided herein.
  • the correct sequence may be isolated by selectively isolating the correct sequence from the other incorrect sequences as by selectively isolating, moving or transferring the desired correct nucleic acid sequence.
  • polynucleotides having an incorrect sequence can be selectively removed.
  • nucleic acid sequences are identical to nucleic acid sequences.
  • a clonal nucleic acid or “clonal population” or “clonal polynucleotide” are used interchangeably and refer to a clonal molecular population of nucleic acids, i.e. to nucleic acids or polynucleotide that are substantially or completely identical to each other. Accordingly, the dilution based protocol provides a population of nucleic acid molecules being substantially identical or identical to each other.
  • the polynucleotides are diluted serially.
  • the device integrates a serial dilution function.
  • the assembly product is serially diluted to produce a clonal population of nucleic acids.
  • the concentration and the number of molecules can be assessed prior to the dilution step and a dilution ratio is calculated in order to produce a clonal population.
  • the assembly product is diluted by a factor of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 10, at least 20, at least 50, at least 100, at least 1,000 etc...
  • the nucleic acid sequences are sorted by an improved limiting dilution technique.
  • the methods use a feedback loop to increase the efficiency of separation beyond the Poisson distribution of limiting dilution.
  • the process disclosed herein is an iterative process. The process can use a fluorescence signal as a feedback mechanism for identification of empty samples, thereby allowing for more effective in vitro cloning than the Poisson limiting dilution.
  • a pool of nucleic acid sequences is diluted to form a diluted solution or mixture of nucleic acid molecules. The nucleic acid molecules are separated into samples comprising a single or a smaller number of molecules.
  • the diluted solution can be used for seeding the wells of a plate such that individual wells of a plate (e.g. 96 well plate) are filled with less than one molecule per well.
  • Cycles of quantitative PCR (qPCR) reactions can be run on all the samples, allowing for identification of samples (e.g. wells) initially seeded with individual molecules. Presence of nucleic acid sequences within each sample can be determined by fluorescence. Because of the way the initial dilution is set, very few samples (e.g. well) would be initially seeded with multiple molecules.
  • step 1 readout of the fluorescence shows the presence of amplification products in a small number of wells (well f, Fig. 1, step 1).
  • Sample seeded which did not contain any nucleic acid molecules can then be supplemented with the dilution solution (step 3, Fig. 1) and quantitative PCR can be performed again.
  • step 4 readout of the fluorescence shows the presence of amplification products in an increasing number of wells (well b, c and f). The process can be repeated until substantially all the samples or wells contain an amplified population of nucleic acid molecules.
  • specific hybridization can be used as a method for identifying and/or retrieving nucleic acids of interest.
  • solid supports such as microparticles or beads, having a single ligand site for attachment of nucleic acid can be used to retrieve a single nucleic acid molecule.
  • the single ligand site can be an oligonucleotide sequence complementary to part of the sequence of the nucleic acid sequences to be analyzed or complementary to an oligonucleotide tag attached to the nucleic acid sequences.
  • Each nucleic acid of the plurality of nucleic acid sequence can be tagged with a different oligonucleotide tag or with the same oligonucleotide tag.
  • the nucleic acid sequences of the plurality of nucleic acid sequences can be ligated to a single oligonucleotide tag.
  • a plurality oligonucleotide tags are provided.
  • each oligonucleotide tag has the same length.
  • the length of the oligonucleotide tag is at least 4 nucleotides long, at least 20 nucleotides long, at least 20 nucleotides long, at least 30 nucleotides long, or at least 40 nucleotides long.
  • solid supports having complementary sequences immobilized thereon are provided.
  • Complementary sequences may be sequences complementary to the oligonucleotide tag sequence or sequences complementary to a portion of the plurality of nucleic acid sequences.
  • Oligonucleotide sequences may be covalently or non- covalently linked to the surface of the solid support.
  • the solid support may be a bead, microparticle, plate, membrane, array and the like.
  • the solid support is a microparticle or a bead.
  • the size range of the bead range from 1 ⁇ to 1000 ⁇ diameter.
  • the size of the beads may be chosen to facilitate the manipulation of the beads.
  • the complementary sequences are attached to the solid support. Yet in other embodiments, the sequences complementary to the oligonucleotide tags are synthesized on the solid support as disclosed herein.
  • the solid support is made of controlled pore glass, nucleoside-derivatized CPG, cellulose, nylon, acrylic copolymers, dextran, polystyrene, magnetic beads and the like.
  • the solid support is porous. Typically, the beads have a pore size ranging from 500 to 1000 angstrom. Yet in other embodiments, the solid support is non porous.
  • a population of beads each bead having thereon a single complementary sequence to the oligonucleotide tag can be used such as each bead of the population is capable of retrieving a single nucleic acid molecule.
  • the nucleic acid molecules are mixed with beads comprising the single ligand site (e.g. single complementary sequence) under conditions favorable for the hybridization complementary sequences.
  • the plurality of beads having nucleic acid sequences attached thereon may be suspended and separated into samples ( e.g. in wells of a microtiter plate, Fig. 2, step 2).
  • the beads may be subjected to limited dilution or to the methods disclosed herein and distributed and separated such as each well contains a single bead. Nucleic acid sequences immobilized onto the isolated beads may be subjected to amplification using primers.
  • the target polynucleotides are amplified after obtaining clonal populations.
  • the target polynucleotide may comprise universal (common to all oligonucleotides), semi-universal (common to at least a portion of the oligonucleotides) or individual or unique primer (specific to each oligonucleotide) binding sites on either the 5 ' end or the 3 ' end or both.
  • the term "universal" primer or primer binding site means that a sequence used to amplify the oligonucleotide is common to all oligonucleotides such that all such oligonucleotides can be amplified using a single set of universal primers.
  • an oligonucleotide contains a unique primer binding site.
  • the term “unique primer binding site” refers to a set of primer recognition sequences that selectively amplifies a subset of oligonucleotides.
  • a target nucleic acid molecule contains both universal and unique amplification sequences, which can optionally be used sequentially.
  • primers/primer binding sites may be designed to be temporary.
  • temporary primers may be removed by chemical, light based or enzymatic cleavage.
  • primers/primer binding sites may be designed to include a restriction endonuclease cleavage site.
  • a primer/primer binding site contains a binding and/or cleavage site for a type lis restriction endonuclease.
  • amplification sequences may be designed so that once a desired set of oligonucleotides is amplified to a sufficient amount, it can then be cleaved by the use of an appropriate type lis restriction enzyme that recognizes an internal type lis restriction enzyme sequence of the oligonucleotide.
  • the pool of nucleic acids may be contacted with one or more endonucleases to produce double-stranded breaks thereby removing the primers/primer binding sites.
  • the forward and reverse primers may be removed by the same or different restriction endonucleases.
  • restriction endonuclease Any type of restriction endonuclease may be used to remove the primers/primer binding sites from nucleic acid sequences.
  • a wide variety of restriction endonucleases having specific binding and/or cleavage sites are commercially available, for example, from New England Biolabs (Beverly, Mass.).
  • a detectable label can be used to detect one or more nucleotides and/or oligonucleotides described herein.
  • detectable label is used to detect amplified molecules, for example, after quantitative PCR.
  • detectable markers include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, protein-antibody binding pairs and the like.
  • fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin and the like.
  • bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, aequorin and the like.
  • enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorimidases, phosphatases, peroxidases, cholinesterases and the like.
  • Identifiable markers also include radioactive compounds such as 1251, 35S, 14C, or 3H. Identifiable markers are commercially available from a variety of sources.
  • the oligonucleotide probes or nucleotides are fluorescently labeled with four different fluorophores, each fluorophore being associated to a particular base or nucleotide.
  • the sequence of one or more nucleic acid molecules is determined and/or the nucleic acid molecules having the correct sequences of interest are selectively isolated. This can typically accomplished by conventional or next generation primary sequencing.
  • methods to sequence verify nucleic acid sequences using high throughput sequencing are provided.
  • nucleic acid molecules are sequenced by synthesis. Sequence determinations can be made by any available method permitting the querying of the sequence of an individual molecule ("single molecule sequencing"), whether directly or through the querying of an amplified population of nucleic acids derived from a single molecule (“polony sequencing"). The method of sequence determination can be non-destructive, to the extent that the objective of the sequence
  • polony amplification can involve, for example, in situ polonies, in situ rolling circle amplification, bridge PCR, picotiter PCR, or emulsion PCR.
  • an oligonucleotide to be amplified is prepared to include primer binding sites, whether as part of its sequence when initially synthesized or by subsequent ligation to adaptor molecules bearing the primer binding sites.
  • the oligonucleotides are immobilized at distinct locations (e.g., predetermined, addressable locations or random locations) on a solid support.
  • the methods involve the use of a library of identifying oligonucleotide tags (also referred herein as barcodes).
  • the length of the oligonucleotide tags may vary depending on the size of complexity of the nucleic acid population to be analyzed.
  • each barcode differs from the other by at least 2 nucleotides. In preferred embodiments, a portion of the library of
  • oligonucleotide tags is used and attached to one end terminus of the plurality of nucleic acid sequences in the population (or pool) in a stoichiometry such that a small sampling (i.e.
  • the nucleic acid population may contain X nucleic acid molecules and a plurality m of nucleic acid tags can be provided wherein m inferior to X.
  • the resulting sub-population will comprise m bar-coded nucleic acid molecules, the remaining nucleic acid molecules being free of
  • oligonucleotide tags ( "unbar-coded") .
  • a number of primers n specific to a known set n of specific barcode sequences can be added, wherein n is inferior to m. Addition of primers specific to the oligonucleotide tags or barcode under favorable conditions results in the hybridization of the primers to their specific biding sites and allows for specific amplification of a subset n of the bar-coded nucleic acid sequences.
  • the barcode or oligonucleotide tags are attached to the end terminus of a sub-population population of nucleic acid sequences thereby forming nucleic acid sequences-oligonucleotide tag conjugates.
  • the barcode or oligonucleotide tag can be attached using TA cloning technique. This technique relies on the ability of adenine (A) and thymine (T) on different nucleic acid sequences (e.g. oligonucleotide tag sequences and nucleic acid sequences of interest) to hybridize and ligate together in presence of ligase.
  • the oligonucleotide tag sequences or the nucleic acid sequences to be analyzed are amplified using a Taq polymerase which preferentially adds a A at the 3 ' end.
  • the sequences comprising a A at the 3' end are hybridized and ligated to sequences comprising a T at the 5 ' end.
  • each barcode sequence is expected to appear only once in the subpopulation, thus ensuring clonality.
  • the bar-coded subpopulation may then be amplified using primers for a known set of specific barcode sequences (i.e. a subset pool), which represents a desired sampling (or subset) of the subpopulation of nucleic acids.
  • this amplified subset population may then be separated into samples
  • primers specific to the individual barcode sequence can be used to preparatively clone an individual molecule from the original population of nucleic acid molecules.
  • Poisson statistics due to the balance of the probability of any specific barcode appearing exactly once as a ligated product to a nucleic acid molecule from the mixture.
  • other manipulations may be performed, for example sequencing the amplified subset population.
  • the attached barcode can be used for multiplex sequencing on a high throughput next generation sequencing
  • the sequences of each nucleic acid molecule from the mixture as well as its attached barcode can be determined.
  • the sequencing data may then be used to determine target molecules of interest from the mixture, as well as the identity of which barcodes they are attached to in the subset pool. These target molecules may then selectively amplified from the subset pool in individual wells, clonally separating them from non-target sequences in the mixture.
  • the present invention provides among other things novel methods and devices for high-fidelity gene assembly. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

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Abstract

L'invention a trait à des procédés et à des dispositifs concernant l'isolement d'acides nucléiques d'intérêt dans une population d'acides nucléiques, telle que des banques de séquences d'acides nucléiques.
PCT/US2012/042597 2011-06-15 2012-06-15 Procédés de préparation de clonage in vitro WO2012174337A1 (fr)

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EP12735365.4A EP2721154B1 (fr) 2011-06-15 2012-06-15 Procédés de préparation de clonage in vitro
CN201280039777.8A CN103732744A (zh) 2011-06-15 2012-06-15 制备性体外克隆的方法
LTEP12735365.4T LT2721154T (lt) 2011-06-15 2012-06-15 Paruošiamojo in vitro klonavimo būdai
AU2012271487A AU2012271487B2 (en) 2011-06-15 2012-06-15 Methods for preparative in vitro cloning
CA2839418A CA2839418A1 (fr) 2011-06-15 2012-06-15 Procedes de preparation de clonage in vitro
EP18207413.8A EP3517611A3 (fr) 2011-06-15 2012-06-15 Procédés de préparation de clonage in vitro
IL230009A IL230009B (en) 2011-06-15 2013-12-17 Methods for preparative in vitro cloning
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US10207240B2 (en) 2009-11-03 2019-02-19 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
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US10207240B2 (en) 2009-11-03 2019-02-19 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
US9968902B2 (en) 2009-11-25 2018-05-15 Gen9, Inc. Microfluidic devices and methods for gene synthesis
US9216414B2 (en) 2009-11-25 2015-12-22 Gen9, Inc. Microfluidic devices and methods for gene synthesis
US11071963B2 (en) 2010-01-07 2021-07-27 Gen9, Inc. Assembly of high fidelity polynucleotides
US9217144B2 (en) 2010-01-07 2015-12-22 Gen9, Inc. Assembly of high fidelity polynucleotides
US9925510B2 (en) 2010-01-07 2018-03-27 Gen9, Inc. Assembly of high fidelity polynucleotides
US11845054B2 (en) 2010-11-12 2023-12-19 Gen9, Inc. Methods and devices for nucleic acids synthesis
US10457935B2 (en) 2010-11-12 2019-10-29 Gen9, Inc. Protein arrays and methods of using and making the same
US11084014B2 (en) 2010-11-12 2021-08-10 Gen9, Inc. Methods and devices for nucleic acids synthesis
US10982208B2 (en) 2010-11-12 2021-04-20 Gen9, Inc. Protein arrays and methods of using and making the same
US11702662B2 (en) 2011-08-26 2023-07-18 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
US10927369B2 (en) 2012-04-24 2021-02-23 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US10081807B2 (en) 2012-04-24 2018-09-25 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US11072789B2 (en) 2012-06-25 2021-07-27 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
WO2016007604A1 (fr) 2014-07-09 2016-01-14 Gen9, Inc. Compositions et procédés pour le clivage et la coupure d'adn dirigés

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AU2012271487A1 (en) 2014-01-16
IL287337A (en) 2021-12-01
AU2012271487B2 (en) 2017-05-25
EP3517611A3 (fr) 2019-09-04
LT2721154T (lt) 2019-04-25
US20220162673A1 (en) 2022-05-26
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US20180023120A1 (en) 2018-01-25
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CN103732744A (zh) 2014-04-16
EP3517611A2 (fr) 2019-07-31

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